Farkas D H, McMahon L W, Cai P, Umek R M
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, MI 48073-6769.
Am J Clin Pathol. 1993 Oct;100(4):444-50. doi: 10.1093/ajcp/100.4.444.
A Southern blot-based assay is presented that increases the simplicity and accuracy of the HER-2/neu gene copy number assignment in breast cancer DNA. Genomic DNA was hybridized simultaneously with probes corresponding to portions of HER-2/neu and a single-copy gene, myeloperoxidase. A unique restriction fragment was detected for each gene. The use of DNA probes of similar mass and equal specific activities resulted in a ratio of band intensities on the resultant autoradiograph that reported the ratio of gene copy numbers directly. Patient samples containing amplified levels of the HER-2/neu gene were identified by simple visual inspection of a single autoradiograph. Analysis of breast cancer samples alongside the cell line DNAs, representing a range of HER-2/neu gene copy numbers, permits visual quantitation of the tumors' gene copy numbers. The authors show that the HER-2/neu gene copy number can be determined accurately in marginally degraded DNA, a feature of some clinical samples.
本文介绍了一种基于Southern印迹的检测方法，该方法提高了乳腺癌DNA中HER-2/neu基因拷贝数测定的简便性和准确性。基因组DNA与对应于HER-2/neu部分和单拷贝基因髓过氧化物酶的探针同时杂交。每个基因检测到一个独特的限制性片段。使用质量相似且比活性相等的DNA探针，使得所得放射自显影片上的条带强度比直接反映基因拷贝数比。通过简单目视检查单一放射自显影片即可鉴定出含有扩增水平HER-2/neu基因的患者样本。将乳腺癌样本与代表一系列HER-2/neu基因拷贝数的细胞系DNA一起分析，可直观定量肿瘤的基因拷贝数。作者表明，在某些临床样本具有的轻微降解的DNA中也能准确测定HER-2/neu基因拷贝数。
